A systematic analysis of the structure, function, and regulation of the pyruvate and alpha-ketoglutarate dehydrogenase complexes from microorganisms and from mammalian tissues is continuing. The mammalian pyruvate dehydrogenase complex is regulated by a phosphorylation-dephosphorylation cycle. Phosphorylation and concomitant inactivation of the pyruvate dehydrogenase component of the complex is catalyzed by a MgATP2-requiring kinase, and dephosphorylation and concomitant reactivation is catalyzed by a Mg2 ion - and Ca2 ion-requiring phosphatase. ADP inhibits the kinase competitively with respect to ATP, provided K ion or NH4 ions are present. Pyruvate inhibits kinase activity noncompetitively with respect to ATP. We have obtained evidence that the interconversion of the active, nonphosphorylated form of pyruvate dehydrogenase and its inactive, phosphorylated form is also modulated by acetyl-CoA/CoA and NADH/NAD molar ratios. An increase in either ratio increases the proportion of the phosphorylated form of pyruvate dehydrogenase. It appears that the kinase activity is stimulated by acetyl-CoA and by NADH and is inhibited by CoA and by NAD. NADH apparently inhibits the phosphatase activity, and this inhibition is reversed by NAD. Experiments are in progress to obtain pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase in a homogeneous state and in adequate amounts for further characterization. Systematic studies will be conducted with peptide substrates to elucidate the effects of modifiers on kinase and phosphatase activities and the specificity requirements of these two regulatory enzymes, and comparative studies will be conducted on the regulatory properties of the kinase and the phosphatase from different animal tissues. BIBLIOGRAPHIC REFERENCES: Pettit, F.H., Pelley, J.W., and Reed, L.J., "Regulation of Pyruvate Dehydrogenase Kinase and Phosphatase by Acetyl-CoA/CoA and NADH/NAD Ratios," Biochem. Biophys. Res. Commun., 65, 575-582 (1975). Reed, L.J., "Regulation of Mammalian Pyruvate Dehydrogenase Complex by Phosphorylation and Dephosphorylation," in Thiamine (C.J. Gubler, M. Fujiwara and P.M. Dreyfus, eds.), p. 19-30, John Wiley & Sons, Inc., New York (1976).